1. Field of the Invention
The present invention relates to apparatus and methods for detection and enumeration of sample particles in translucent or transparent flowing liquid. In particular the present invention relates to high throughput analysis of imaged particles in a translucent flow.
2. Description of the Prior Art
Detection and enumeration of low concentrations of selected microorganisms in large volumes of fluid has a number of applications including: 1.) bioterrorism and biowarfare defense, 2.) food and water quality control, 3.) clinical detection of pathogens, and 4.) environmental monitoring. Biodetection systems developed to date usually suffer from 1.) high cost, 2.) unsatisfactory sensitivity, 3.) slowness 4.) large size and/or 5.) labor-intensive preparation steps.
Common biodetection techniques include culture-based techniques, molecular microbiological techniques (such as Polymerase Chain Reaction or PCR), direct detection using epifluorescent microscopy, conventional flow cytometry and solid phase cytometry. All of these techniques suffer drawbacks. Culture-based techniques are slow (typically taking more than 24 hours) and usually require labor intensive steps. Microbiological techniques are not strictly quantitative and are limited by interference from background material. PCR, in particular, doesn""t differentiate between live and dead cells. Direct detection of single cells without the requirement for growth is desirable, as it is more rapid. In addition, detection is quantitative, as the number of cells is directly determined.
A conventional technique for direct enumeration of microorganisms in water is filtration and microscopy. A known water sample volume is filtered so that cells in the size range of the microorganisms of interest are captured. These are then stained and examined by microscopy. Typically, staining of specific microorganisms is obtained through use of immunofluorescent antibodies (IFAs). IFAs are fluorescent molecules chemically bonded to antibodies. The antibodies are produced to have binding specificity for particular target microorganisms or particular cell surface receptors. Hence, staining a target cell with an IFA specific for it causes the cell to fluoresce and xe2x80x9cstand outxe2x80x9d from background, unstained material when illuminated. Filters are examined under epifluorescence microscopy. When the organism(s) of interest are specifically stained, they fluoresce against the background. In order to retain organisms as small as bacteria, the filter used for capture must have a small pore size, typically 0.22-0.45 microns. The volume filtered is limited to several liters maximum as membranes quickly clog with particulates. Microscopic scanning for bacteria is labor intensive, requiring a trained microscopist and a large non-portable epifluorescence-equipped microscope.
Direct detection may also be accomplished using flow cytometry. Flow cytometry is a commonly used technique to measure the chemical or physical properties of cells. Cells flow by a measuring apparatus in single file while suspended in a fluid, usually air or water. In immunofluorescence flow cytometry, cells can be identified by attaching fluorescent antibodies to each cell:
An antibody specific to the cell of interest is labeled with a fluorescent molecule or fluorochrome.
The labeled antibody is mixed in solution with the cell of interest. The antibodies attach to specific sites on the cells (called antigens).
The cells are passed in single file in a stream of liquid past a laser(s), which illuminates the fluorochromes and causes them to fluoresce at a different wavelength.
A photomultiplier or photodiode is used to detect a burst of fluorescence emission each time a marked cell passes in front of the detector.
The number of marked cells can then be counted. Antibodies can be chosen that are highly-specific to the cell(s) of interest.
Flow cytometry is currently used for a wide variety of applications including: measuring helper T-lymphocyte counts to monitor HIV treatment, measuring tumor cell DNA content in determining cancer treatment, and separating X- and Y-chromosome bearing sperm for animal breeding.
FIG. 1 (prior art) shows a typical flow cytometry system (from Shapiro, Practical Flow Cytometry, 2nd Edition). Putting flow cytometry into practice involves using two concentric cylindrical streams of fluid. The inner flow or core flow contains the cells to be sampled. The purpose of the outer stream or sheath flow is to reduce the diameter of the core flow. As the core and sheath fluids reach the tapered region of the flow, the cross-sectional area of the core flow is reduced. A small bore core flow (xcx9c20 xcexcm) allows for precision photometric measurements of cells in the flow, illuminated by a small diameter laser beam; all of the cells will pass through nearly the same part of the beam and will be equally illuminated. Why not just pass the cells through a small-bore transparent tube? Small diameter orifices are generally unworkable because they experience frequent clogging. All commercial flow cytometers now use a sheath/core flow arrangement.
Laser-induced fluorescence of fluorescent labels in a flow cytometer is a uniquely powerful method of making fast, reliable, and relatively unambiguous detections of specific microorganisms, such as foodborne pathogens. Several monographs describe the methods and applications of flow cytometry (e.g., Flow Cytometry: First Principles by A. L. Givan, 1992, and references therein). The successful detection of single cells relies on several critical factors. First, the laser power must be sufficient to generate a large enough number of fluorescence photons during the brief passage of the labeled microbial cell through the irradiated region. Specifically, it is essential that the number of photons generated be large enough so that the fluorescence burst can be reliably differentiated from random fluctuations in the number of background photons. Second, reducing the background is important, i.e., minimizing the number of unwanted photons that strike the detector which arise from scattering and fluorescence of the apparatus and of impurities and unbound dye in the flowing liquid.
Historically, flow cytometers have been very large, expensive laboratory-based instruments. They consume large amounts of power, and use complex electronics. They are not typically considered within the realm of portable devices. The size (desktop at the smallest), power requirements, and susceptibility to clogging (requiring operator intervention) of conventional flow cytometers precludes their use for many applications, such as field monitoring of water biocontamination.
A technique called solid-phase cytometry is more rapid than flow cytometry. In this method, the fluid matrix is concentrated and then filtered. Fluorescently labeled microorganisms are then imaged on a filter membrane with a laser scanning system, detected, and enumerated. Using fluorescently tagged antibodies, this technique can detect specific microorganisms, and is highly suited for low concentrations of microorganisms. However, the high cost of this system ( greater than $100,000) makes its use problematic for many applications, especially those requiring continuous on-site monitoring.
U.S. Pat. No. 6,309,886, xe2x80x9cHigh throughput analysis of samples in flowing liquid,xe2x80x9d by Ambrose et al. is an invention for the high throughput analysis of fluorescently labeled DNA in a transparent medium. This invention is a device that detects cells in a flow moving toward an imaging device. The flow is in a transparent tube illuminated in the focal plane from the side by a laser with a highly elongated beam. Although this invention does not suffer from the drawbacks listed above for alternative technologies, it is not suitable for applications where the flow medium is not transparent.
A need remains in the art for improved apparatus and methods for high throughput analysis of samples in a translucent flowing liquid.
The present invention has the advantages of solid-phase cytometry, including detection of individual microorganisms, at a much lower cost ( less than $10,000). This invention is based on taxonomic identification using fluorescent dyes, including immunofluorescent dyes. The detection steps include:
An antibody specific to the cell species of interest is labeled with a fluorescent molecule or fluorochrome.
The labeled antibody is mixed in solution with the cell species of interest. The antibodies attach to specific sites on the cells (antigens).
The cells are passed in a vertical stream of liquid toward a laser diode, gas laser, or the like, which illuminates the fluorochromes and causes them to fluoresce at a longer wavelength.
A low-cost CCD (charge coupled device) 2-D detector detects a flash of fluorescence emission each time a marked cell is illuminated by the laser diode while passing through the focal plane of the detector.
The number of marked cells is then counted via computer. Antibodies can be chosen that are highly specific to the cells of interest.
One object of the present invention is to provide apparatus and methods for high throughput, high sensitivity detection and identification of samples in a translucent or transparent flowing liquid. This object is accomplished by providing a relatively large cross section axial flow, in which cells or other target particles suspended in a liquid are observed as they emerge from a tube oriented generally along the optical axis of imaging optics.
The present invention includes apparatus and methods for imaging multiple fluorescent particles in a sample passing through a flow channel. A flow channel defines a flow direction for samples in a flow stream and has a viewing plane nearly perpendicular to the flow direction. A clear volume between the illuminated flow volume and the imaging optics is provided in some embodiments. A beam of illumination is formed as a column having a size that can effectively cover the viewing plane, and illuminates the flow nearly end on, or from the side. Imaging optics are arranged to view the focal plane to form an image of the multiple fluorescent sample particles in the flow stream. A camera records the image formed by the imaging optics.
In one embodiment, optics are provided for multiple emission wavelength dispersion, including a multi-segmented, motorized filter wheel.
The present invention includes apparatus and methods for for identifying particles in a sample of liquid flowing within a flow channel along a flow axis comprising a transparent element transverse to the flow axis for terminating the flow channel and diverting the sample, a waste conduit adjacent to the transparent element for discharging the sample, an illumination beam positioned to illuminate an illumination zone within the sample, and an imaging element for imaging a focal plane transverse to the flow axis within the illumination zone.
Generally the focal plane is spaced apart from the transparent element and a clear volume separates the transparent element and the imaging element.
The imaging element comprises a color filter, optics, and a CCD camera.
The waste conduit may comprise an opening formed in the flow channel, or a specially designed flow block. The flow block includes an input tube for providing the sample to the flow block, an output tube for discharging the sample from the flow block, and a flow path formed within the flow block and connected to the input tube and the output tube. The flow path passes adjacent to the transparent element, and the focal plane is located in the flow path before the sample passes adjacent to the transparent element.
The illumination may be provided by a laser or by an LED.
The invention may further include a multicolor detection assembly comprising a generally planar filter wheel located between the transparent element and the imaging element, the filter wheel plane tilted with respect to the optical axis of the imaging element, wherein the filter wheel includes at least two filter portions, each filter portion having a different thickness or index of refraction, and a filter wheel motor for rotating the filter wheel such that illumination from the illumination zone passes through each filter portion and is imaged by the imaging element during part of the integration time. Spaced apart images of each imaged particle are then formed for each filter portion.
In the case of detection of pathogenic microorganisms in ground beef, the resulting bacteria/extract mixture would contain, blood, fat, and tissue, and would likely be optically thick over circa 1 mm or greater distance necessitating a transparent gap (air or immersion liquid) between imaging optics and the focal plane of the instrument.
In this invention, as a microorganism passes up the delivery tube its spot size (the size that the image of the microorganism occupies on the CCD) decreases as it approaches the focal plane of the CCD camera; above the focal plane the spot size increases. The spot size reaches it minimum in the focal plane of the CCD fore-optics. In order to maximize the signal to noise ratio, the spot size must be minimized. This occurs when an exposure is made while the microorganism (or fluorescent target particle) is passing through the focal plane perpendicular to the optical axis of the CCD camera.